6024541NCGCNS17012013319qHTS Assay for Inhibitors of Influenza NS1 Protein Function: Viral Replication TCID50Influenza is a world-wide public health problem and emerging forms of the virus have the potential to cause a pandemic of equal or greater magnitude to the outbreaks recorded in 1918, 1957 or 1968. Vaccine development is proceeding and there also exist two classes of anti-influenza compounds. However these therapeutic modalities are neither fully effective nor widely enough available to fulfill global needs. In addition their potential usefulness against newly emergent strains is not known. Efforts are needed to develop novel agents against influenza virus, including broad-spectrum agents. Identification of small molecules that inhibit NS1 function either directly or by interfering with specific cellular pathways may be a key to increasing our defense against the virus. We have miniaturized and optimized a cell based assay in which NS1 from influenza A is expressed in the budding yeast S. cerevisiae. NS1 is a multi-functional protein that counters the host innate immune response and facilitates viral versus cellular gene expression. Expression of NS1 causes a pronounced slow growth phenotype in yeast due to its intrinsic molecular activities. Small molecules that suppress the slow growth phenotype can be identified by a straightforward growth recovery assay using optical density (OD) as the measurement. The same yeast strain not expressing NS1 was used as positive control in the yeast growth recovery assay.Hits resulting from the primary screen and other follow-up screens, were tested in two concentrations in this viral replication assay.NIH Chemical Genomics Center [NCGC]NIH Molecular Libraries Probe Centers Network [MLPCN]MLPCN Grant: MH084878Assay Submitter (PI): Daniel Engel, University of Virgina Cell monolayers were infected at an m.o.i. of 0.1 for 48 h in the presence or absence of the compound, which was added at the beginning of infection. Virus titres were determined by TCID50 analysis (Reed, L. J. & Muench, H. (1938). A simple method of estimating fifty per cent endpoints. Am J Epidemiol 27, 493-497)Compounds that showed over 8 fold replication at 10 uM are considered "active"; below 2-fold at 10 uM are considered "inconclusive", and 0-fold at 10 uM are considered "inactive"."Active compounds are assigned a score of 90, "inconclusive" 50, and "inactive" 10.2336Summary956533NS1 nonstructural protein NS1 [ Influenza A virus ]4932http://www.ncgc.nih.gov19052087http://en.wikipedia.org/wiki/Virus_quantification1TCID50 at 5 uM12542TCID50 at 10 uM12543Compound QC42541nonstructural protein 1 [Influenza A virus (A/WSN/1933(H1N1))]1943523801Influenza A virus (A/WSN/1933(H1N1))taxon3828350MH084878212488870012901928.663QC'd by "NCGC"2012321124888706129019293QC'd by "NCGC"2012321124888792129018.527.663QC'd by "NCGC"2012321124951240129018.8828.683QC'd by "NCGC"2012321124951241129018.8828.53QC'd by "NCGC"2012321124951242135018.527.663QC'd by "NCGC"2012321124951244135018.3327.663QC'd by "NCGC"2012321124951245129019293QC'd by "NCGC"201232112495124612901928.53QC'd by "NCGC"201232112495124712901928.663QC'd by "NCGC"201232112495124812901928.333QC'd by "NCGC"2012321124954142129019293QC'd by "NCGC"201232112495414312901928.663QC'd by "NCGC"201232112495414412901928.663QC'd by "NCGC"2012321124954145135018.527.333QC'd by "NCGC"2012321124954146135027.333QC'd by "NCGC"2012321124954147129018.528.53QC'd by "NCGC"201232112495414812901928.663QC'd by "NCGC"2012321124954149135018273QC'd by "NCGC"2012321124954150129018.527.663QC'd by "NCGC"2012321124954151129018.6628.663QC'd by "NCGC"2012321124954152129017.66263QC'd by "NCGC"2012321124954153129019293QC'd by "NCGC"2012321124954154129018.527.663QC'd by "NCGC"2012321124954155129018.6628.53QC'd by "NCGC"2012321124954156135018263QC'd by "NCGC"2012321124954157129019293QC'd by "NCGC"2012321124954158135018.6627.333QC'd by "NCGC"2012321124954159129019293QC'd by "NCGC"2012321124954160135018.33263QC'd by "NCGC"2012321124954161129019293QC'd by "NCGC"201232112495416212901928.333QC'd by "NCGC"2012321124954163129018.6628.663QC'd by "NCGC"2012321124954164129018.6628.333QC'd by "NCGC"2012321124954165129018.6628.663QC'd by "NCGC"2012321124954166129018.6628.333QC'd by "NCGC"2012321124954996129019293QC'd by "NCGC"2012321124954997129019293QC'd by "NCGC"2012321124954998135018.3326.53QC'd by "NCGC"2012321124954999135018.33263QC'd by "NCGC"2012321